Neurodegeneration can be triggered by a variety of genetic, epigenetic, and environmental factors. Healthy neurons are able to maintain their integrity throughout the life of an animal, suggesting the existence of a maintenance mechanism that allows neurons to sustain, mitigate or even repair damage. Previous work in our lab and others has found NMNAT proteins in Drosophila and mammals to be robust and versatile neuroprotective factors. However, it remains unclear whether and how neurons regulate the neuroprotective processes mediated by maintenance factors such as NMNAT. How neurons partition NMNAT into two distinct functions - housekeeping (NAD synthesis) and neuroprotection, and how such partitioning is regulated under normal and adverse conditions to achieve neuroprotection are the foci of this proposal. Our preliminary data indicate that Drosophila nmnat gene is alternatively spliced into two mRNA variants (RA and RB) that lead to two sets of functionally distinct proteins: PA/PC, with higher enzyme activity, and PB/PD, with enhanced chaperone function. We hypothesize that post-transcriptional regulation, including alternative splicing, functions as a switch between the neuroprotective and enzymatic roles of NMNAT, and that neurons use this switch mechanism to regulate the neuroprotective response under normal and disease conditions. We propose to characterize the hypothesized switch mechanism in Drosophila and then extend the study to human NMNATs in mammalian neurons. First, we will characterize the biochemical and cellular properties of Drosophila and human NMNAT protein variants, and illustrate the role of alternative splicing in post- transcriptional regulation of nmnat genes. Next, we will identify alternative splicing as a stress response that affords protection to neurons, and further reveal the functional role of microRNAs in regulating the abundance of neuroprotective RNA variants and modulating the neuroprotective efficacy of NMNAT. Finally, we will study human NMNAT protein variants and characterize the neuroprotective function of alternatively spliced NMNAT variants in cultured DRG explants and in vivo in cortical spinal track neurons, and identify key mechanisms underlying the divergent neuroprotective effects of NMNAT variants in degenerative conditions. Studies on Drosophila and human NMNATs - a two-model approach - will reveal the evolutionarily conserved regulatory mechanisms and identify strategies relevant to enhancing neuroprotection in humans.